Identification of lysine decarboxylase as a mammalian cell growth inhibitor in Eikenella corrodens: possible role in periodontal disease.

The pathogenesis of inflammatory periodontal disease was studied by examining the mechanism of HeLa and HL60 cell growth inhibition by cell-free saline-soluble extracts of Eikenella corrodens and bacterial plaque. Previous studies identified a protein (p80) as causing growth inhibition by E. corrodens extracts. After purification by two-dimensional SDS-PAGE, p80 was digested with protease lysC. Amino acid sequences were obtained and backtranslated for use as PCR primers. A 5840 nucleotide sequence containing a lysine decarboxylase gene was obtained from a Sau3 A1 genomic library of E. corrodens DNA. Lysine decarboxylase activity was present at physiologic pH in the E. corrodens extracts containing p80, and also in bacterial plaque. Both extracts caused growth inhibition by depleting lysine from cell culture media through conversion to cadaverine. Adding lysine, or immune goat IgG to a peptide derived from the active site sequence of E. corrodens lysine decarboxylase, retarded lysine depletion and growth inhibition. epsilon-Amino caproic acid specifically enhanced lysine decarboxylase activity at the low lysine concentration in HL60 cell culture media, and also increased the growth inhibition. Thus, lysine decarboxylases such as p80 inhibit growth by removing lysine from mammalian cell culture media. A new role for lysine decarboxylase activity in the microbial aetiology of periodontal disease is discussed.

Impact of adolescent pregnancy as we approach the new millennium.

Between 1990 and 1997 teen pregnancy and birth rates have fallen dramatically, especially in black teens. However, the teen birth rate in the United States remains the highest of any industrialized nation: four times higher than Germany, six times higher than France. Contraception use has also increased. Condoms were used during last intercourse approximately 55% of the time in 1997 vs. only approximately 38% in 1991. Likewise, the percentage of teens having intercourse has dropped, and the number of abortions is down. The negative social and economic impact of early teenage pregnancy is tremendous. It is estimated to cost the nation about $21 billion annually (in 1993 dollars). The long-term productive life prospects are also lower for teenage mothers and their offspring. Many programs have been utilized to reduce the incidence of teen pregnancy. While some have had modest success, no single or simple solution is on the horizon.

Maternal mortality in Kentucky.

This paper reviews maternal mortality in Kentucky over the 30 year period of 1966 through 1995. Data were reviewed from the minutes of the State Maternal Mortality Study Committee and the reports of the State Cabinet of Health Services, State Centers for Health Statistics. There has been a marked fall in the number of births during this 30 years and an even more dramatic reduction in maternal mortality. Identifiable causes for maternal death have changed over the years. Today, the three leading causes of maternal mortality in Kentucky mirror those of the nation, i.e., hemorrhage, pulmonary embolus, and toxemia.

An Eikenella corrodens toxin detected by plaque toxin-neutralizing monoclonal antibodies.

Bacterial plaque from the gingival region of teeth contains cytotoxic agents which lyse undifferentiated human HL60 cells. A small panel of monoclonal antibodies (MAbs) was found to abrogate much of this activity and to detect antigens in certain strains of Streptococcus mitis and Eikenella corrodens. The aim of this study was to determine whether these bacterial antigens might be involved in HL60 cells cytolysis. Saline extracts were obtained by homogenizing washed, stationary-phase cells in 65 mM NaCl with a tight-fitting Potter-Elvehjem homogenizer. The extracts of E. corrodens were toxic to HL60 cells, whereas similar extracts of S. mitis were nontoxic. Adding plaque toxin-neutralizing MAb 3hE5 blocked the toxic effect of E. corrodens extract S. mitis extracts contained a single, strongly reactive antigen of 140 kDa (s140K antigen) detected on Western blots (immunoblots) by three MAbs from the panel. Rabbit antibodies raised to this antigen excised from the gel (anti-s140K serum) detected larger antigens in addition to s140K. E. corrodens extracts contained a number of antigens detected by the MAbs. Immunoglobulin G (IgG) was purified from anti-s140K serum by passage through DE52 cellulose. A 100-fold excess (by weight) of the purified IgG to E. corrodens protein specifically cross-precipitated an 80-kDa antigen plus a nonantigenic 16-kDa protein, presumably attached noncovalently. The remaining supernatant fraction had no toxic activity. A similar ratio of control IgG (from nonimmunized rabbits) did not precipitate these proteins, and the supernatant fraction had the same activity as the extract not treated with IgG. The proteins of 80 and 16 kDa were also detected in the anti-s140K immunoprecipitate by rabbit IgG antibodies to E. corrodens whole cells. The 80-kDa antigen, alone or complexed with the 16-kDa protein, may be involved in mediating the toxic activity in E. corrodens and plaque extracts.

Pulmonary hypertension associated with long-term inhalation of "crank" methamphetamine.

We present the case of a 33-year-old white male truck driver with a ten-year history of 3.5-g/wk "crank" and "peanut butter methamphetamine" inhalation (methamphetamine and propylhexedrine, respectively). This patient developed marked pulmonary hypertension as a probable consequence of abuse of these drugs. Proposed mechanisms of disease invoke toxic endothelial injury, hypoxic insult, direct spasm, vasculitis, and dysregulation of mediators of vascular tone. The possible role of genetics is discussed in reference to variability of human response to sympathomimetic challenge.

New monoclonal antibodies identify the glycoprotein carrying the CA 125 epitope.

CA 125 is an antigenic determinant located on the surface of ovarian carcinoma cells and elevated in the serum of greater than 90% of patients with carcinoma. The antigen, derived from the ovarian epithelium, has been described as a mucinlike glycoprotein greater than 200 kd. To date little is known of the metabolic regulation or expression of this antigen in either normal or neoplastic tissues. New monoclonal antibodies that we describe here recognize both unique and similar epitopes to OC 125. These reagents may allow for a more complete definition of the structure and expression of the CA 125 complex. These antibodies recognize high-molecular weight (greater than 200 kd) subspecies and a lower-molecular-weight (68 kd) subspecies of the antigen and identify it in the cytoplasm and the extracellular matrix of CA 125-producing cells.

Use of monoclonal antibodies with neutralizing effects on toxic antigens from human bacterial plaque to detect specific bacteria by colony blotting.

Inflammatory periodontal diseases are provoked by bacteria which adhere to teeth at the gingival margin and form plaques containing toxins detectable by their effect on mammalian cells in culture. The aim of this study was to make toxin-neutralizing monoclonal antibodies and determine whether they detect antigen in specific oral bacteria. Bacterial plaque was collected from teeth and homogenized, and the fluid phase (plaque extract) was boiled or first fractionated over Sephacryl S-300. Hybridomas from immunized mice secreted immunoglobulin M (IgM) antibodies which reacted to plaque antigens. Neutralization was detected by an increase in the growth of HL60 cells which were exposed to plaque toxins in the presence of IgM from hybridoma culture or ascitic fluids. However, the neutralization was obvious only when the plaque toxins reduced growth by 50% or less. Plaque toxin preparations were found to contain proteases which hydrolyzed all of the IgM in ascitic fluids within 24 h. Replenishing the IgM daily preserved protection compared with protection from IgM from other hybridomas or saline only. The decrease in the specific activity of plaque proteins caused by replenishing one such antibody (3hE5) was 2.5-fold compared with activity with unreplenished 3hE5, 3.8-fold compared with activity with saline only, and 10.7-fold compared with activity with replenished, unrelated antibody. The neutralizing IgM detected an array of 14,000- to 22,000-molecular-weight antigens. The native toxins may be aggregates of these antigens, or the array may indicate fragments of an undetected, larger antigen or a common, nonpeptide adduct. Only 0.5 to 0.8% of the bacteria from sites with periodontitis and grown on blood agar contained antigen. One group of reactive bacteria was identified as Actinomyces odontolyticus serotype I. Other isolates were identified as Staphylococcus epidermidis, but antigen disappeared from the these isolates within 6 weeks of subculture. Epitope-containing antigens were also found in streptococcal and Eikenella isolates, and it is likely that the antigens from only some of these bacteria are toxic.

The N-terminal and C-terminal portions of the atrial natriuretic factor prohormone increase during preeclampsia.

The influence of preeclampsia on the circulating concentrations of the 28-amino-acid carboxy terminus (C-terminus) (i.e., atrial natriuretic factor) and the amino terminus (N-terminus) of the 126-amino-acid atrial natriuretic factor prohormone (pro ANF) was studied in the third trimester with the use of three specific radioimmunoassays that recognize: (1) atrial natriuretic factor (i.e., amino acids 99 to 126), (2) the whole 98-amino-acid N-terminus, and (3) amino acids 31 to 67 from the midportion of the N-terminus of the prohormone. The C-terminus was significantly increased (p less than 0.001) in the third trimester in women with preeclampsia, the mean +/- SEM of 15 subjects was 150 +/- 7 pg/ml versus 89 +/- 7 pg/ml in the third trimester in 12 women during normal pregnancies and 65 +/- 2 pg/ml in 19 healthy nonpregnant women. The whole 98-amino-acid N-terminus, likewise, was significantly increased (p less than 0.001) in women with preeclampsia to 4706 +/- 629 pg/ml versus 2160 +/- 79 pg/ml in women in the third trimester of normal pregnancies and versus the circulating concentration of 1847 +/- 127 pg/ml in healthy nonpregnant women. ProANF 31 to 67 mean circulating concentration in preeclampsia was 4638 +/- 725 pg/ml, which was also significantly (p less than 0.001) increased compared with its mean circulating concentration in the third trimester of normal pregnancy of 1758 +/- 83 pg/ml or that in healthy nonpregnant women (1400 +/- 105 pg/ml). The circulating concentrations of both the N-terminus and C-terminus of the atrial natriuretic factor prohormone decreased within 24 hours after delivery in contrast to a normal pregnancy in which they both increase post partum. These results indicate a marked difference in the metabolism of both the N-terminus and the C-terminus of the atrial natriuretic factor prohormone in women with preeclampsia versus that in women with normal pregnancies or that in healthy nonpregnant women.

Determination of plutonium 240/239 ratios in Lake Ontario sediments.

A thermal ionization mass spectrometric procedure was developed for determination of 240/239 ratios in subpicogram quantities of plutonium (Pu), and was applied to Pu extracted from Lake Ontario sediment. The detection limit was ∼ 4 fg of Pu and the precision of 240/239 ratio measurement was ∼ 7% in the 0.07-0.42 pg range. Results of sediment analyses at various depths showed in all cases 240/239 ratios near the mean global fallout value of 0.176 ± 0.014. The lack of variability of the ratios with depth negated the use of Pu, present in excess in the near-surface samples due to leakage from a nearby reprocessing plant, as a geochronological meter. The 240/239 ratio of the leakage Pu appeared to be very similar to that of fallout Pu.

Distribution of CA 125 in embryonic tissues and adult derivatives of the fetal periderm.

New murine monoclonal antibodies to a partially purified CA 125 antigen were developed and identified as M 2 and M 11. With immunohistochemical techniques, these new antibodies and OC 125 antibody were used to search for CA 125 in embryonic tissues and adult apocrine sweat glands and mammary glands. The embryonic skin, the periderm, expressed CA 125 antigen and its adult derivatives, the mammary glands and apocrine sweat glands, expressed CA 125 while in the active state of secretion. In a 6-week-old formalin-fixed and paraffin-embedded ectopic embryo specimen, antibodies M 2 and M 11 recognized CA 125 in the periderm, the notochord, the myocardium, the pericardium, the gastroenteric tract, enteric duct remnants in the umbilical cord (vitelline and allantoic ducts), the mesonephric duct, and the amnion. OC 125 staining of these formalin-fixed specimens was either very faint or absent. In a formalin-fixed and paraffin-embedded specimen of apocrine sweat glands from the axilla, antibodies M 2 and M 11 detected CA 125 antigen intracellularly in the secretory cells. Again no staining was observed with OC 125 antibody. In a frozen and acetone-fixed specimen of lactating mammary glands, antibodies M 2 and OC 125 detected CA 125 antigen intraductally. Colostrum and milk collected from 25 mothers at various stages post partum had mean CA 125 levels of 34,213 U/ml in colostrum, 1469 U/ml at 3 to 7 days, and 105 U/ml at 5 to 26 weeks.

Oxygen desaturation in women in labor.

Oxygen saturation and end-tidal CO2 tension were monitored in 15 healthy women during labor. Oxygen saturation was determined with a pulse oximeter and end-tidal CO2 with a CO2 monitor. Fetal heart rate, uterine contractions and maternal blood pressure were also monitored. End-tidal CO2 tension was followed to determine if falls in oxygen saturation during labor were related to hyperventilation. Ten of the 15 patients exhibited periods of oxygen saturation of less than 90%. End-tidal CO2 was consistently low, usually less than 30 mm Hg. Most, but not all (7 of 10), of the patients who showed desaturation had received narcotics. There were often periods of apnea and/or shallow respirations between contractions. These aberrations and hyperventilation-induced hypocarbia were probably the cause of the oxygen desaturation. No changes in fetal heart rate or low Apgar scores were noted.

Increase in the plasma levels of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor during normal pregnancy.

The influence of pregnancy on the circulating concentrations of atrial natriuretic factor, the 28 amino acid carboxy (C)-terminal end of the 126 amino acid atrial natriuretic factor prohormone, and the amino (N)-terminus of the prohormone was studied with three specific radioimmunoassays recognizing: (1) atrial natriuretic factor (i.e., amino acids 99 through 126), (2) the 98 amino acid N-terminus, and (3) amino acids 31 through 67 from the midportion of the N-terminus of the prohormone. Plasma atrial natriuretic factor in normal pregnant women gradually increased as pregnancy progressed, with the mean +/- SEM being 58 +/- 4 pg/ml in the first trimester, 74 +/- 5 pg/ml in the second trimester, and 89 +/- 7 pg/ml in the third trimester. Likewise, proatrial natriuretic factor 31 through 67 increased from 1421 +/- 76 pg/ml (first trimester) to 1509 +/- 84 pg/ml (second trimester) to 1758 +/- 83 pg/ml in the third trimester, whereas the whole N-terminus of the prohormone increased from 1804 +/- 98 pg/ml (first trimester) to 1909 +/- 111 pg/ml (second trimester) to 2160 +/- 79 pg/ml in the third trimester. These results suggest that release of the N-terminus of the prohormone, as well as atrial natriuretic factor, increases with the rise in blood volume associated with a normal pregnancy. The circulating concentrations of both the C-terminus and N-terminus of the atrial natriuretic factor prohormone increased further in the 48 hours after delivery. Because both the C-terminus and N-terminus of the atrial natriuretic factor prohormone contain diuresis-producing peptides, these results suggest that postpartum diuresis may be mediated by these peptides.

Physical modeling of the composting ecosystem.

A composting physical model with an experimental chamber with a working volume of 14 x 10 cm (0.5 ft) was designed to avoid exaggerated conductive heat loss resulting from, relative to field-scale piles, a disproportionately large outer surface-area-to-volume ratio. In the physical model, conductive flux (rate of heat flow through chamber surfaces) was made constant and slight through a combination of insulation and temperature control of the surrounding air. This control was based on the instantaneous conductive flux, as calculated from temperature differentials via a conductive heat flow model. An experiment was performed over a 10-day period in which control of the composting process was based on ventilative heat removal in reference to a microbially favorable temperature ceiling (temperature feedback). By using the conduction control system (surrounding air temperature controlled), 2.4% of the total heat evolved from the chamber was through conduction, whereas the remainder was through the ventilative mechanisms of the latent heat of vaporization and the sensible temperature increase of air. By comparison, with insulation alone (the conduction control system was not used) conduction accounted for 33.5% of the total heat evolved. This difference in conduction resulted in substantial behavioral differences with respect to the temperature of the composting matrix and the amount of water removed. By emphasizing the slight conduction system (2.4% of total heat flow) as being a better representative of field conditions, a comparison was made between composting system behavior in the laboratory physical model and field-scale piles described in earlier reports. Numerous behavioral patterns were qualitatively similar in the laboratory and field (e.g., temperature gradient, O(2) content, and water removal). It was concluded that field-scale composting system behavior can be simulated reasonably faithfully in the physical model.

Matric water potential as an ecological determinant in compost, a substrate dense system.

Water is a critical ecological factor in substrate dense matric ecosystems, of which composting systems are an example. Excessive moisture inhibits aerobic metabolism because of oxygen diffusion limitations. How a lack of water impedes composting activity has not been previously considered. An investigation of the relationship of matric water potential (water held by physical attractions) to gravimetric water content was carried out in a laboratory composting system using a sewage sludge and wood chip substrate. The gravimetric-matric water relationship was best fit by a 2∘ polynomial regression of y=64.049-0.142 x, andR= 0.95. It is proposed that the commonly observed failure of composting to occur under drier conditions is a physical limitation, that is bacteria progressively fail to physically colonize the substrate as matric potential decreases below approximately -20 kPa. In physically mixed systems, agitation and introduction of inoculum may promote colonization, permitting composting to be initiated and proceed at lower moisture contents. In nonmixed systems, promotion of thorough initial colonization may permit composting to continue in the presence of significant drying.

The use and interaction of permanent pacemakers and the automatic implantable cardioverter defibrillator.

The adverse interactions of permanent pacemakers and automatic implantable cardioverter defibrillators (AICD) were studied in nine patients in whom both devices were implanted. Both unipolar and bipolar pacemakers were evaluated. The permanent pacemakers were also used to do noninvasive electrophysiological studies and to induce ventricular fibrillation. Undersensing of ventricular fibrillation by the permanent pacemakers caused inappropriate pacemaker stimuli, which caused undersensing of ventricular fibrillation by the AICD in three of four patients with unipolar pacemakers. After an AICD discharge, pacemaker noncapture was seen in eight of 22 episodes for an average 4.9 seconds and inability to sense was seen in 11 of 20 episodes for an average 9.0 seconds. Counting of pacemaker stimuli and QRS by the AICD caused inappropriate discharges. Noninvasive electrophysiological testing by the pacemakers correlated with invasive testing. Furthermore, induction of ventricular fibrillation was successful in four of five patients attempted, though requiring long bursts at high outputs at the shortest cycle lengths obtainable by these pacemakers. Operation of the AICD and permanent pacemakers must be clearly understood to avoid adverse interactions of these devices.

Noninvasive electrophysiologic study using standard permanent pacemakers in patients with spontaneous sustained ventricular tachycardia or ventricular fibrillation.

Permanent pacemakers capable of noninvasive electrophysiologic testing were used to study and treat 26 patients with spontaneous sustained ventricular tachycardia (VT) or fibrillation (VF). One hundred nine episodes of sustained VT or VF were induced in these patients. In 8 patients spontaneous VT was reverted by noninvasive means. Drug changes based on noninvasive testing were made in 12 patients. In the 1- to 67-month follow-up period, drug therapy based on noninvasive electrophysiologic testing was predictive of outcome in patients with spontaneous arrhythmias. Thus, noninvasive electrophysiologic testing using permanent pacemakers is a useful method for studying and treating patients with recurrent sustained ventricular arrhythmias.

Ventricular arrhythmias during reperfusion.

Accelerated idioventricular rhythm has been used as a marker for coronary reperfusion. The incidence of accelerated idioventricular rhythm and ventricular tachycardia was evaluated in 52 consecutive patients undergoing thrombolysis with intracoronary streptokinase during acute myocardial infarction. Complete 12-hour Holter recordings during and after intracoronary streptokinase were obtained in 39 patients. Reperfusion was documented in 17 patients (44%), no reperfusion in 14 (36%), and subtotal occlusion in eight (20%). Accelerated idioventricular rhythm occurred in 83%, 57%, and 63% of patients by group, respectively (p greater than 0.05). Ventricular tachycardia occurred in 100%, 71%, and 100% of patients by group, respectively (p less than 0.05). These data demonstrate that accelerated idioventricular rhythm is not specific for reperfusion and cannot be used as a marker for this event, and that ventricular tachycardia is more common with reperfusion and subtotal occlusion.